Fluorescence microscopy is a light microscopy technique for studying the structure or properties of a sample by imaging fluorescent or phosphorescent emission from target species, such as organic molecules or inorganic compounds, located on or in a sample. For instance, a sample may be labeled with fluorophores, molecules that absorb light around an excitation wavelength and, in response, fluoresce, emitting light at an emission wavelength typically longer than the excitation wavelength. A fluorescence image of the sample is obtained by detecting the emitted fluorescence.
Total internal reflection fluorescence (TIRF) microscopy is a technique that probes fluorescence only near the surface of a sample. In TIRF, total internal reflection of incident light occurs when a light beam impinges on a sample at or greater than a critical angle, which depends at least in part on optical properties of the sample and the surrounding environment. Although the incident light is reflected, an evanescent wave having the same wavelength as the incident light is generated in the sample. The evanescent wave decays exponentially away from the surface of the sample and penetrates only a small depth into the sample. Thus, only fluorophores near the surface of the sample are excited. Because only a small region of the sample is probed by TIRF, the signal-to-noise ratio of the resulting image is improved relative to standard fluorescence microscopy techniques.